Studies carried out during the current grant period have provided additional insights on two different mechanisms of bimodal targeting of xenobiotic inducible cytochrome P450s (CYPs) to mitochondria and microsomes (ER): 1) a pathway involving endoproteolytic cleavage of nascent proteins, which applies to CYP1A1 and possibly other members of family 1 CYPs. 2) a pathway involving PKA mediated phosphorylation at internal sites as shown for CYP2B1,2E1,3A1/2 and possibly other members family 2. An endoprotease capable of processing CYP1A1 at the +33 position has been purified and the site of mitochondrial-imported +33/1A1 (CYPMT2) interacting with Adrenodoxin has been mapped. Based on this the major objectives are to continue to elucidate mechanistic details of the two pathways with special focus on the characterization of endoprotease and phosphorylation mediated conformational changes at the signal and Adx binding regions of CYP2E1, and undertake new studies on the physiological functions of mitochondrial 2E1 and human mitochondrial 2D6 as follows: 1). The purified cytosolic endoprotease will be further characterized by sequencing, cloning and by overexpression in mammalian cells. The role of this protease in mitochondrial targeting of other family 1 CYPs, such as 1B1 and non-CYP proteins will be tested. 2). Studies on mechanism of mitochondrial targeting of CYP2E1 will be continued with a focus on altered conformation of N-terminal signal domain, mode of interaction with Adx, and altered substrate specificity. It is proposed to investigate the role of mitochondrial CYP2E1 (designated as CYPMT5) in mitochondrial drug metabolism, ROS production and mitochondrial toxicity using stable cell lines that express either microsomal 2E1 or mitochondrial MT5.3). Mechanisms of mitochondrial targeting of human CYP2D6 and genotype-specific changes that affect mitochondrial targeting will be investigated. Stable cell lines expressing WT and mutant CYP2D6 will be used to investigate the physiological consequence of vastly reduced mitochondrial CYP2D6 in human livers on drug metabolism, drug induced toxicity, and mitochondrial function. [unreadable] [unreadable] [unreadable]